Eukaryotic proteins are synthesized within the endoplasmic reticulum (ER), are delivered from the ER to the Golgi complex for post-translational processing and sorting, and are transported from the Golgi to specific intracellular and extracellular destinations. This intracellular and extracellular movement of protein molecules is termed vesicle trafficking. Trafficking is accomplished by the packaging of protein molecules into specialized vesicles which bud from the donor organelle membrane and fuse to the target membrane.
Specialized cell types utilize specific vesicle trafficking routes. For instance, in endocrine glands, hormones and other secreted proteins are delivered to secretory granules for exocytosis through the plasma membrane to the cell exterior. In macrophages, peroxidases and proteases are delivered to lysosomes. In fat and muscle cells, glucose transporters are stored in vesicles which fuse with the plasma membrane in response to insulin stimulation.
Numerous proteins are necessary for the formation, targeting, and fusion of transport vesicles and for the proper sorting of proteins into these vesicles. The vesicle trafficking machinery includes coat proteins which promote the budding of vesicles from donor membranes; vesicle- and target-specific identifiers (v-SNAREs and t-SNAREs) which bind to each other and dock the vesicle to the target membrane; and proteins which bind to SNARE complexes and initiate fusion of the vesicle to the target membrane (SNAPs).
Vesicles in the process of budding from the ER and the Golgi are covered with a protein coat similar to the clathrin coat of endocytotic vesicles. The protein coat is assembled from cytosolic precursor molecules and is confined to budding regions of the organelle membrane. The coat protein (COP)-coated vesicles are uncoated after budding is complete to allow fusion of the vesicle to the donor membrane.
The COP coat consists of two major components, a guanosine triphosphatase (GTPase) and coat protomer (coatomer). The individual proteins are cytosolic until assembled into the protein coat. Coatomer is an equimolar complex of seven proteins, termed .alpha., .beta., .beta.', .gamma., .delta., .epsilon., and .zeta.-COP. Two types of COP vesicles have been characterized, COPI and COPII, which contain different GTPase components (Rothman, J. E. et al. (1996) Science 272:227-234). The GTPase subunit, ARF protein in COPI coats and the closely-related SAR protein in COPII coats, controls the budding process. In a process essentially the same for the SAR/COPII complex, ARF binds GTP and inserts into the organelle membrane, which initiates COPI assembly. Membrane-bound ARF-GTP recruits cytosolic coatomer proteins. Co-assembly of ARF and coatomer into the COPI coat on the organelle membrane surface drives vesicle budding. Subsequent hydrolysis of the bound GTP induces vesicle uncoating by releasing ARF and coatomer. COPI vesicles are involved in protein trafficking from the ER to Golgi, bidirectional movement within the Golgi, and from Golgi to the ER. COPII vesicles are involved primarily in ER to Golgi trafficking.
Subunits of coatomer have been cloned from a variety of mammalian sources. The 36 kdal .epsilon.-COP has been cloned from bovine liver (Hara-Kuge S. et al. (1994) J. Cell. Biol. 124:883-892) and from hamster (Guo, Q. et al. (1994) J. Cell. Biol. 125:1213-1224). Examination of a conditional lethal, temperature-sensitive (ts) .epsilon.-COP mutant of chinese hamster ovary (CHO) cells indicates that .epsilon.-COP and associated COPI coatomers are necessary for the establishment or maintenance of Golgi structure, for proper ER-to-Golgi transport of integral membrane and secreted proteins, and for normal endocytotic recycling of low-density lipoprotein (LDL) receptors (Guo, et al. 1994).
The etiology of numerous human diseases and disorders can be attributed to defects in the trafficking of proteins to organelles or the cell surface. Defects in the trafficking of membrane-bound receptors and ion channels are associated with cystic fibrosis (cystic fibrosis transmembrane conductance regulator; CFTR), glucose-galactose malabsorption syndrome (Na.sup.+ /glucose cotransporter), hypercholesterolemia (low-density lipoprotein (LDL) receptor), and forms of diabetes mellitus (insulin receptor). Abnormal hormonal secretion is linked to disorders including diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone; ACTH).
Cancer cells secrete excessive amounts of hormones or other biologically active peptides. Disorders related to excessive secretion of biologically active peptides by tumor cells include: fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal cramps, diarrhea, and valvular heart disease, caused by excessive amounts of vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) secreted from intestinal tumors. Ectopic synthesis and secretion of biologically active peptides (peptides not expected from a tumor) includes ACTH and vasopressin in lung and pancreatic cancers; parathyroid hormone in lung and bladder cancers; calcitonin in lung and breast cancers; and thyroid-stimulating hormone in medullary thyroid carcinoma.
Polynucleotides encoding a novel human coatomer vesicle protein and the molecules themselves provide a means to investigate vesicle trafficking and secretion under normal and disease conditions. Discovery of a novel coatomer vesicle protein satisfies a need in the art by providing new compositions useful in diagnosing and treating disorders associated with abnormal vesicle trafficking.